Aspartate transcarbamoylase (ATCase) is encoded by the pyrBI genes in E. coli. Expression of these genes is reduced four-fold by attenuation when grown on uracil. Using plasmid, pRO1727. the pyrB and the pyrBI genes from E. coli were cloned into a P. putida pyrB auxotroph. A recombinant pyrB gene was recovered that encoded a functional hybrid ATCase with a molecular weight of 470 kDa.

Enterohemorrhagic Escherichia coli (EHEC) are important foodborne pathogens with a bovine reservoir. For many years, research and regulations have focused on the EHEC serogroup most commonly associated with severe human illness, EHEC O157. However, six additional EHEC serogroups have been identified as important human foodborne pathogens and have been declared adulterants in raw, non-intact ground beef by the United States Department of Agriculture’s Food Safety Inspection Service. Collectively these seven organisms are referred to as EHEC-7. With the addition of these six pathogens, epidemiological studies are needed to estimate the probability for cattle to carry them and to identify risk factors associated with their presence in samples of bovine origin. In addition, the potential for pre-harvest control of EHEC-7 in feedlot cattle, particularly by dietary intervention, is a knowledge gap that needs to be addressed. Finally, detection methods of EHEC-7 have not been validated, and there is no “gold-standard” test. The first study included in this dissertation was a cross-sectional study estimating the prevalence and risk factors associated with hide contamination by EHEC-7 on the hides of market beef cows at slaughter. The second study was a longitudinal analysis of EHEC-7 from fecal samples from cow-calf herds in Mississippi and Nebraska. The third study was a randomized controlled trial evaluating the effects of fiber from distillers grains on the probability to detect EHEC-7 in samples from the rectoanal mucosa of feedlot steers. The fourth study included in this dissertation was a Bayesian latent class analysis estimating the diagnostic performance of three EHEC-7 detection methods, specifically modeling the performance and prevalence of EHEC O157 in fecal samples from beef cow-calf herds.

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Strains of Escherichia coli may exist as a beneficial human commensal or a pathogen capable of causing morbidity and mortality. Of the E. coli which causes human disease, many strains which cause bacteremia have been identified as possessing virulence factors which make them more resistant to the complement system. The bacterial amyloid fibril, curli, functions in bacterial adherence and the formation of biofilm. Curli-producing parental and curli-deficient mutant E. coli was compared in its survival to human complement, using in vitro serum sensitivity assays. Results showed an increase in the survival of curli-producing E. coli, which suggested that curli defends against complement killing. An in vivo murine model of E. coli-induced sepsis demonstrated that curli-producing bacteria also survived significantly better in the blood of mice. Immunostaining and flow cytometry was done to determine if parental and mutant strains of E. coli differentially bind to complement components C1q and C3. Results demonstrated that curli increases binding of C1q, but does not affect C3 binding. Blocking the classical pathway suggested that, in these assays, the classical pathway was the major contributor to complement activation and curli inhibits its activity. In addition, blocking the alternative pathway supported that the classical pathway was the main mechanism for complement activation and suggested that curli is not involved in protecting E. coli against alternative pathway activation. Results of this study conclude that curli defends E. coli against complement killing via inhibition of the classical complement pathway. / Microbiology and Immunology